弱激光照射对小鼠骨髓来源巨噬细胞极化的调控作用  

The modulation of low-level laser on polarization of mouse bone marrow-derived macrophages

作　　者：戴晨[1] 宋基伟[1] 梁卓文[1] 张倩[2] 张坤[2] 王哲[1] 胡学昱[1] 

机构地区：[1]第四军医大学西京骨科医院,陕西西安710032 [2]第四军医大学神经科学研究所,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2016年第8期1045-1050,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81070996)

摘　　要：目的研究不同能量参数的810 nm弱激光照射对于巨噬细胞极化状态的影响。方法体外分离、培养BALB/c小鼠来源的骨髓巨噬细胞,应用巨噬细胞集落刺激因子(M-CSF)条件性培养基培养的骨髓细胞,流式细胞术检测F4/80表达鉴定培养结果。脂多糖-γ干扰素(LPS-IFN-γ)刺激进行M1细胞极化诱导,反转录PCR检测诱导型一氧化氮合酶(i NOS)、精氨酸酶1(Arg1)、CD86 mRNA水平,Western blot法检测i NOS和Arg1蛋白水平。应用(1、2、3、4)J/cm2的810 nm弱激光对体外培养的M1细胞进行照射,MTT法检测细胞增殖,免疫荧光细胞化学染色检测i NOS、Arg1的表达,反转录PCR检测M1细胞i NOS、Arg1 mRNA水平,Western blot法检测M1细胞i NOS、Arg1蛋白水平。结果流式细胞术结果证明分离细胞的F4/80阳性率达99.9%。骨髓源性巨噬细胞在LPS-IFN-γ刺激下,高表达i NOS和CD86 mRNA。Western blot法检测发现,给予极化刺激后,骨髓源性巨噬细胞高表达i NOS和CD86蛋白。不同能量的弱激光照射M1型巨噬细胞,MTT法检测发现,(1、2、3)J/cm2弱激光刺激时,M1细胞活力与对照组比较无显著性差异;4 J/cm2刺激时,M1细胞活力降低。免疫荧光细胞化学染色显示,与对照组比较,(1、2)J/cm2照射时,M2型巨噬细胞标志物Arg1阳性细胞数无明显差异,照射剂量为(3、4)J/cm2时,Arg1阳性细胞数显著增多,M1型巨噬细胞标志物i NOS阳性细胞数显著减少。与对照组比较,照射剂量为(1、2)J/cm2时,i NOS及Arg1 mRNA和蛋白表达水平无明显变化,在照射剂量为(3、4)J/cm2时,i NOS mRNA和蛋白水平表达下降,Arg1 mRNA和蛋白水平表达升高。结论 (1、2)J/cm2的810 nm弱激光对于M1型巨噬细胞活力及极化无影响。照射剂量为3 J/cm2时,M1型巨噬细胞可向M2型巨噬细胞极化,且细胞活性无明显变化。但照射能量为4 J/cm2时,M1型细胞虽然可向M2型细胞极化但同时伴有细胞活性降低。Objective To investigate the influence of 810 nm low-level laser of different energy on the polarization of macrophages. Methods The macrophages were isolated from the bone borrow of BALB/c mice and cultured in macrophage colony stimulating factor （M-CSF） conditioned cultural medium. The expression of F4/80 was examined by flow cytometry for identification. After lipopolysaccharide-y interferon （LPS-IFN-y） induced polarization status in the macrophages, the mRNA expressions of inducible nitric oxide synthase （iNOS）, arginase 1 （Argl） and CD86 were detected by reverse transcription PCR, and the protein expressions of iNOS and Arg] were tested by Western blotting. Thereafter, the M1 macrophages were exposed to 810 nm low-level laser of （1, 2, 3, 4） J/cm2, and then the cell viability was evaluated by MTT assay; the expressions of iNOS and Argl were observed by immunofluorescent cytochemical staining; the mRNA and protein levels of iNOS and Argl were studied by reverse transcription PCR and Western blotting. Results Flow cytometry showed that the percentage of F4/80 positive cells cultured with M-CSF conditioned medium was 99.9%. The mRNA and protein levels of iNOS and CD86 in macrophages were both significantly raised after induction by LPS-IFN-y. Compared with the control ceils, the viability of MI cells significantly decreased when the energy of the low-level laser exposure was 4 J/cm2, while the viability remained unchanged when the energy was t, 2 or 3 J/cm2. Immunocytochemistry revealed that the Dercentaqe ofArgl positive cells that represent M2 macrophages was not significantly different from the control group when the irradiation dose was t or 2 J/cm2, however, the Arg] positive cells significantly increased and the iNOS positive cells that represent M1 macrophages significantly decreased when the irradiation dose was 3 or 4 J/cm2. When the irradiation dose was 1 or 2 J/cm2, the mRNA and protein levels of iNOS and Argl remained unchanged compared with the control group. When the irradiatio

关 键 词：810 nm弱激光 脊髓损伤 M1/M2细胞极化 骨髓来源巨噬细胞 

分 类 号：R744[医药卫生—神经病学与精神病学]